Vulnerability to mental illness and ability to cope with stress are powerfully shaped by individual differences in human temperament and personality. Understanding the mechanisms whereby biological and environmental factors interact to shape brain development, temperament, and vulnerability to stress and emotional dysfunction is crucial for generating improved preventative treatments for a variety of psychiatric disorders, such as major depression and anxiety. To elucidate molecular and neuroanatomical changes in the developing brain that lead to a highly fearful, anxious, and stress-vulnerable phenotype, we developed an advantageous rat model that may permit analysis of biological mechanisms of individual differences in temperament. To do so, we selectively-bred rats for differences in emotional reactivity, and combined this with molecular and epigenetic profiling. Our High-Responder (HR) rats vigorously explore novel environments and exhibit greater impulsivity, aggression, and risk-taking versus Low-Responder (LR) rats, which are very inhibited and show high levels of spontaneous anxiety and depressive-like behavior (i.e. immobility in the Forced Swim Test, diminished sexual interest, and anhedonia). Such HR/LR traits are heritable, but are also sensitive to early-life environmental factors, including naturally-occurring variation in HR v. LR maternal style. Using genome-wide expression profiling, we discovered dramatic gene expression differences in the developing hippocampus, amygdala, and prefrontal cortex of HR vs. LR rats, including changes in genes involved in metabolism and synaptic plasticity. These findings suggest that the distinct HR/LR behavioral phenotypes involve molecular changes that drive differential establishment of hippocampal-limbic circuits. Furthermore, our preliminary data suggest epigenetic differences (in HR/LR DNA methylation patterns) may elicit these differences in gene expression and ultimately behavior. The proposed work will use cutting-edge next-generation sequencing to map DNA methylation patterns (the methylome) in the developing and adult brain of HR and LR animals to (a) interrogate inborn methylome differences that may drive their distinct behavioral phenotypes; (b) determine how an early-life experience (cross-fostering) reconfigures the developing LR neural methylome to influence behavior; and (c) test whether manipulating DNA methylation in the HR/LR brain suffices to shift their phenotypes. We will thereby test our working hypothesis that HR/LR methylome differences constitute a key phenotype-driving molecular mechanism by testing whether manipulating methylation in the HR/LR brain modifies their behavior. This work will illuminate how epigenetic mechanisms may drive individual differences in emotionality, stress vulnerability, and risk for emotional dysfunction. Unraveling such complicated genetic, epigenetic, neurobiological, and environmental interactions in a model organism of temperamental and stress vulnerability/resilience differences should yield important results relevant to understanding the developmental neurobiology of mood disorders and how to develop improved treatments.